R. equi Infection
A significant endemic pathogen that affects the equine breeding industry is the facultative bacterial pathogen, Rhodococcus equi. The intracellular bacteria are part of the mycolic acid-containing group of actinomycetes, which includes Corynebacterium, Mycobacterium, and Nocardia (Embley et al., 1994). R. equi is an opportunistic bacterium that is abundant in soil and herbivore manure and takes advantage of the underdeveloped immune system of 1-6 month old foals (Giguere et al., 1997). This can lead to pyogranulomatous bronchopneumonia, resulting in death of the foals if not treated (Giguere et al., 1997). Foals, sheep, pigs, goats, cattle and immunocompromised humans are all vulnerable to infection by R. equi, with foals being the primary host (Prescott, 1991). Infected swine and cattle develop pyogranulomatous adenitis, whereas other species develop pneumonia and pulmonary lesions, followed by a disruption of the bronchial and mesenteric lymph nodes, which is similar in infected foals (Prescott, J F 1991; von Bargen and Haas, 2010). Foals are infected with R. equi when they inhale aerosolized bacteria particles from contaminated feces, dust, or from the exhalations of contaminated foals; R. equi multiplies in the intestine and disseminates into the environment through fecal matter (Muscatello et al., 2007; Muscatello et al., 2009; von Bargen and Haas, 2010). Alveolar macrophages phagocytize inhaled R. equi and form an R. equi containing vacuole (RCV), documented to block the phagosome-lysosome fusion, inhibit acidification and prevent any further maturation of the vacuole (Zink et al., 1987; Fernandez-Mora et al., 2005). The RCV environment allows R. equi to multiply and survive, resulting in necrotic death of the macrophage and the formation of granuloma as R. equi proliferates (Toyooka et al., 2005; Lührmann et al., 2004).
Immune Response to R. equi 
Research suggests that foals are susceptible to R. equi and cannot clear an infection due to their underdeveloped innate, humoral and cell mediated immune responses in comparison to adult horses. R. equi is cleared with a combination of interferon (IFN) gamma-activated macrophages and opsonizing antibodies enhanced by CD4+ T cells (Cauchard et al., 2004; Dawson, 2010). However, it has been demonstrated that foals have a lag synthesis of IgGb antibodies and low levels of IFN gamma that induce an intracellular response (Sheoran et al., 2000; Holznagel et al, 2003; 2000; Breathnach et al., 2006; Ryan et al., 2010). The waning of maternal antibodies and the low levels of IgGb, which are important factors in combating bacterial and viral pathogens, may also contribute to the susceptibility of foals to R. equi (Hines and Hitela, 1996; Wagner, 2006; Dawson et al., 2010). To clear an R. equi infection a T-helper (TH1) cell-mediated response characterized by IFN gamma induction is required, yet due to an immature immune system, foals mount a T-helper (TH2) cell mediated response characterized by IL-4 induction, which is detrimental to the foals and may lead to the development of clinical pneumonia (Giguere et al., 1999 (5041-5047); Breathnach et al., 2006; Dawson et al., 2010; Ryan et al., 2010). Other limiting factors that increase R. equi susceptibility in foals include decreased killing activity of neutrophils, deficient toll-like receptors on macrophages and Cytotoxic T lymphocytes (CTLs) (Dawson, 2010). Foals have low Major Histocompatibility class II (MHC II) expression and CD lb levels by antigen-presenting cells, which also play an important role in the clearance of pathogens in adult horses (Dawson, 2010).
Treatment Against R. equi 
A combination of lipophilic antimicrobial agents can be used as therapy for an extended period of time to treat R. equi infected foals because they have the ability to penetrate cells and target intracellular pathogens (Prescott, 1991). One common treatment combination consists of rifampin and erythromycin but in the last few years, resistance to this combination has been rising and prolonged antimicrobial therapy should be avoided (Buckley et al., 2007; Takai et al., 1997). The use of rifampin or erythromycin alone is not recommended because the efficacy of erythromycin in an intracellular environment is poor, and the combination of the two optimizes protective effects (Nordmann et al., 1992; Prescott and Sweeney, 1985). In recent years macrolides such as, azithromycin and clarithromycin, have replaced erythromycin in the antimicrobial combination as they have less adverse side effects and are administered less frequently (Giguere et al, 2004). Resistance to macrolides is a problem that has surfaced. When resistance to one macrolide occurs the organism is usually resistant to many other macrolides (Cohen, 2006). Another method to prevent an R. equi infection is administration of R. equi-specific hyper immune plasma (HIP) to foals. HIP provides all-around antibodies against R. equi and other factors that induce an immune response, however, it is reported to have variable results (Martens et al., 1989; Hurley and Begg, 1995; Higuchi et al., 1999; Giguere et al., 2002; Perkins et al., 2002). Although the exact component of HIP that provides protection in unknown, VapA and VapC antibodies are claimed to be critical, in addition to other immune components (Hooper-McGrevy et al., 2001; Lopez et al., 2002). Antibiotics have helped treat R. equi infections but they are not a long-term solution due to antibiotic resistance and side effects.
Human Susceptibility to R. equi 
An R. equi infection is rare in healthy humans and only present in immunocompromised patients with symptoms such as cavitated bronchopneumonia, commonly accompanied by fever, dyspnea, lung lesions, chest pain and a nonproductive cough (Cinque et al., 2011; Prescott, 1991). Infection of R. equi in AIDs patients occurs when CD4+ helper T cells are less than 100 cells/μl (Muscatello, 2012). As of 2010, approximately 34 million people live with HIV/AIDS globally and HIV/AIDS resulted in about 1.8 million deaths in 2010 (WHO HIV/AIDS Data and Statistics, 2010). The first R. equi infection in a human was reported in 1967 and only 15 additional cases were reported until 1983 (Golub et al., 1967; van Etta et al., 1983; Eiguchi et al., 2009; Petrosillo et al., 2010). There has been a steady increase of R. equi infections since 1983, primarily due to the increase in HIV infection, organ transplantations and cancer treatments (Weinstock and Brown, 2002). By 2010, more than 200 cases of R. equi infection in immunocompromised individuals were reported worldwide (Topino et al., 2010; Yamshchikov et al., 2010). Human infection occurs via the same route in humans as in foals (Schlusselhuber et al., 2011). Among HIV-infected patients, the mortality rate due to R. equi is within a 50-55% range and within a range of 20-25% among non-HIV immunocompromised patients (Weinstock and Brown, 2002; Schlusselhuber et al., 2011).
Human Treatment Against R. equi 
Introduction of highly active antiretroviral therapy (HAART) in the 1990's has decreased the mortality rate of HIV patients infected with R. equi, with some reporting a survival rate of 100% (Torres-Tortosa et al., 2003; Yamshchikov et al., 2010). No standard treatment for R. equi in humans exists but treatment with a combination of at least two antibiotics is commonly used. R. equi is susceptible to: rifampin, carbapenems, aminoglycosides, glycopeptides, floroquinolones, macrolides, tygecicline and linezolid (El Karoui et al, 2009; Russo et al., 2010; Ferretti et al., 2011). Treatment with antibiotics over time has led to higher doses required for treating HIV-infected people because some R. equi strains are showing resistance to commonly used antibiotics, including penicillins (Weinstock and Brown, 2002).
R. equi Plasmids
The phylogenetically diverse Rhodococcus genus consists of pathogenic species that affect plants or animals and non-pathogenic species that are found in the environment. R. equi carry different types of plasmids with genes that aid in the survival of both the environment and hosts (Bell et al., 1998; Gürtler et al., 2004). Virulent R. equi strains carry an 80-90 kb plasmid critical for virulence, intracellular replication, and survival (Takai et al., 1991; Tkachuk-Saad and Prescott 1991; Giguere et al., 1999). Loss of the plasmid results in an avirulent strain unable to replicate in the macrophage (Hondulas et al., 1994; Giguere, et al., 1997). The virulence plasmid is divided into four regions: replication and partition, conjugation, pathogenicity island (PAI), and an unknown area (Takai et al., 2000; Letek et al., 2008). Polymerase chain reaction (PCR) analysis demonstrates that plasmidless non-pathogenic R. equi strains are common in the environment with virulent plasmids organized into three different categories, vapA+, vapB+, or vapAB-negative, based on the presence of the virulence associated protein genes, vapA and vapB. Research demonstrates that vapB+ plasmids were found in pig isolates, vapA+ plasmids were found in horse isolates and vapAvapB-plasmids were found in bovine isolates (Oldfield et al., 2004; Ocampo-Sosa et al., 2007). All three different plasmids were detected in human isolates with vapA and vapB not occurring on the same plasmid (Ocampo-Sosa et al., 2007). Both vapA+ plasmids and vapB+ plasmids have a conserved housekeeping backbone and a PAI that encodes different vap genes, which are upregulated when the organism invades the macrophage (Letek et al., 2008). The PAI in vapB+ plasmids consists of six full length vap genes: vapB, -Jy -K1, -K2, -L, and vapA+ plasmids consist of six full length vap genes: vapA, -C, -D -E, -G, -H, and three vap pseudogenes vapF, -I, -X (Takai et al., 2000; Letek et al., 2008). The VapA protein has varying percent identity to the other Vap proteins (Table 1; FIG. 1), with the highest percent identity to the VapB protein, also observed in Letek et al, 2008. The VapC protein also shows varying percent identity to the other Vap proteins (Table 1; FIG. 1), with the highest percent identity to the nonfunctional protein, VapF, also observed in Letek et al., 2008. Phylogenetic analysis of the Vap amino acid sequences demonstrates that VapA and VapB are most closely related, in comparison to the other vaps, and share a common ancestral yap gene (Letek et al., 2008).
TABLE 1Comparison of full length R. equi VapA and VapC protein sequencesagainst other full length R. equi Vap proteins:PercentPercentamino acidamino acidsequencesequenceNCBI Accessionidentity withidentity withR. equi VapSEQ IDnumber of R. equiR. equiR. equiproteinNo:Vap proteinVapAVapCA1NP_06676510058B2YP_0021496017555C3NP_06676758100D4NP_0667685543E5NP_0667724048F6NP_0667735065G7NP_0667554656H8NP_0667593955I9ADI502495222J10YP_0021495924056K111YP_0021495955354K212YP_0021495985354L13YP_0021495974454M14YP_0021495995658X15—4347Sequences were analyzed using the BLAST algorithm.Results expressed as a dash (—) indicate that there is not a curated sequence available from NCBI.
SEQ ID No. 1:MKTLHKTVSKAIAATAVAAAAAMIPAGCANATVLDSGSSSAILNSGAGSGIVGSGSYDSSTTSLNLQK DEPNGRASDTAGQEQQYDVHGDVISAVVYQRFHVFGPEGKVFDGDAGGLTLPGAGAFWGTLFTNDLQR LYKDTVSFQYNAVGPYLNINFFDSSGSFLGHIQSGGVSTVVGVGGGSGSWHNA SEQ ID No. 2:MMKALHKTVSRAIAAIATAAAAVLAVAPASVANAAVLDSGGGSALLKDGAGSGEVGSQAYDSSTVSSN LQKAETNGPVGLAGTAEQEQQYDVHGNVISAAVYQKFHVYGPEDMVFDGDAGGLTIPGAGAFWGTLFT SDLQRLYKDTVSFQYNALGTYLNINFFDSSGGFLGHIQAGAVSAVVGVGGGSGSWHNWEVA SEQ ID No. 3:MFRVGRPSKSIAVVASVLCFLALGGTARANVVAPSAWGGAQSAADKEGEGVTLGGVGVLRPHNKDADE QYVHGVVVSALFYNHLRISVDGGMTFDGDGGGLSTPGGGALWGTLTTSDLQQLYDETASFECNAVGPY LNINFYDSYGRILASVQAGGVSTMIGIGGGNGRWHLV SEQ ID No. 4:MVRARAFGRLFTFLLAVAVIATVSMGGANAQELAGTKTSDAALLSGNKAAIPEDKEYDVSGRVVSALV YQYFIVTVDDAEDKKGKTFQGDAGGVTIPGVDFFWGTLHTPDLEKLYSDTVSFQYNAAATFLNINFFD SKGERLGYVLAGAAGTVSGIGGGTGGWE SEQ ID No. 5:MTTVHKKASKAIAFTVALRLPFAGTAVALVLIALTIVAAPTGIAGAREIGAQAWPASQLESGLAVSGN PVGVHDVRMAVHDDSTHTREFKEDDSEKQYPVHGFASSFIFYQTVSIIIDDDGRGGPGKTFEGEAGGI TTPGAAGYAGVLFTSDLERLYRETVSFEYNAVGPYLNINLFAGDGGLLGHVQSGAISSLVGIGGGTGA WR SEQ ID No. 6:MIEYAWYGPSIQSNRCCGDCPILLALGGHRTCRLATPSAWVGTPSAAGKVLPPINNNADEQYAVHGVV FSAVFYNHVRISVDGGMTEDGEGGGLSTPGGGALWGNLMTSDLLCSSYTTKLRRSNVIWPVSKDQLLR QLWWHSWECSRERC SEQ ID No. 7:MSVRTLLAATLVAGISVLAPAGIANAETSMVSTTAASSVEHAANTYDFAEAKSGSSIPAKVAAEQANS YSVHGLVTSLAVYQHFSLTVEGGGKTFTGDSGGISIPGVAVLEGTLFTEDLQHLYSDTVSFEYNAVGP YLNINFFDSHGTLLGHVQSGSIGTVSGIGGGTGGWQ SEQ ID No. 8:MNLSKTTRKFLSRTAVPATFVMALTVPWGCAAPPPLPDGPTHDLPTWREEGANYSDGTMLVRASSNFL EPSTHSDSGQQQWTVQGVLASALVYQRLKLNVEGGETFEGYAGGLSEPGGAMVWGTLFTDNIQRLYDR TESFEFNAVGPYLNVNFFDGHSAILGHAQLGGVSSVIGIGGGTGTWIGDVA SEQ ID No. 9:MPIALTAVALPAGMASAQEMGDHAWSGSRAESDVAVLGKAESAHDDPSLRTPKLKKSNSGNQYRYTVL LSSFIFYQTLSI SEQ ID No. 10:MNLAHVTRKFLVSTAVPVTLVIAFAAPFQFSAPLASAATSDLSIRRDGSAHYSDSTLSLRASSDSPEP TTHGAQQQWAVHGVLASALVYQLLTLTVDGGEQFQGYAGGVSFPGGAAVWGTLFTDDIQRLYDQTASF QFNAVGPYLNVNFFDRHGTLLGHAQLGGVSSVIGIGGGSGTWTGDVA SEQ ID No. 11:MGNARRSWVKAAAAATLTAAAVMVPAGLANAQPLDVGGSSTVVANDAFGSVSLGGHGSSGYGSSSDYG SSSDYDGSGSGFGTAPDVRSQVAASLDEEQQYDVKGDVWSALVYQQFHVEGPQGKVEDGQAGGLTIPG AGAFWGTLFTSDLNRLYADTSSFQYNAVGPYLNINFFDGNGVLLGHIQAGAVSTVTGVGGGTGSWS SEQ ID No. 12:MGNARRSWVKAAAAATLTAAAVMVPAGLANAQPLDVGGSSTVVANDAFGSVSLGGHGSSDYGSSSDYG SSSDYDGSGSGFGTAPDVRSQVAASLDEEQQYDVKGDVWSALVYQQFHVEGPQGKVFDGQAGGLTIPG AGAFWGTLFTSDLNRLYADTSSFQYNAVGPYLNINFFDGNGVLLGHIQAGAVSTVTGVGGGTGSWS SEQ ID No. 13:MRPQSSYRPYVRAIFAAALVAGISILGATGVVNAETSMASNAATSTVHRVAKTCDSNLSENDHSSAET NGQLSFATEATAEQGYTYSVHGLVTSLAVYQHFSLTVEDDGKTFTGDSGGISVTGVAVLKGTLFTEDL QRLYNDTVSFQYNAVGPYMNINFFDSHSTLLGHVQSGSIGTLTGIGGGTGGWR SEQ ID No. 14:MIRTVVGWGAFVLAFSILATGAAYAHAQELEPGGSFSEGILQRNFPLEGEFASVSEPGSGNVSASKVG EESNFAVRGVVVSALFYQHLEITVSGGETEDGDGGGLSVPGGGALWGTLFTRDLQRLYDETVSFEFNA AGLFVNVNFFDKDGILLGHVESGAVSTAVGIGGGTGRWHIV SEQ ID No. 15:RLYDETGPFDFNAAGLFMNVDHFGYRAExpression of Vaps
Environmental signals such as temperature, pH, magnesium and iron concentration, and/or oxidative stress are documented to regulate the expression of most vap genes on the PAI of equine isolates. VapA, an essential virulence protein of R. equi found in equine isolates, is a 15-17 kDa surface expressed lipoprotein linked to the cell wall through a lipid-modified N-terminal which is regulated by pH and temperature (Takai et al., 1991 (439-443); Takai et al., 1992 (2995-2997); Tan et al., 1995; Giguere et al., 1999; Jain et al., 2003). The function of vapA is unknown, however it is thought to help prevent fusion between the RCV and lysosomes (Fernandez-Mora et al., 2005; Toyooka et al., 2005; von Bargen et al., 2009(5676-5681)). Expression of vapA is highly induced by macrophage related stress, and reported that its deletion prevents R. equi replication in macrophages without affecting macrophage attachment (Benoit et al., 2002; Jain et al., 2003; Jacks et al., 2007). Transcription of vapA is regulated by two genes in the PAI: virR, a LysR-type regulator, and orf8, a two component response-like regulator; the absence of either gene decreases virulence of R. equi (Takai et al., 2000; Ren and Prescott, 2003; Russell et al., 2004). Although vapA has been claimed to be essential for virulence, both vapA and the plasmid have been reported to be necessary for virulence in foals (Jain et al., 2003; Giguere et al., 1999). VapB, present in non-equine isolates, is reported to be a surface expressed 20 kDa lipoprotein with a similar structure to vapA (Takai et al., 2000 (71-80); Byrne et al., 2001; Letek et al., 2008). Plasmids that carry vapB are less virulent than plasmids that encode vapA and are categorized as intermediately virulent (Takai et al., 1996; Takai et al., September 2000).
VapC, a secreted protein, and VapA are reported to induce the strongest lymphoproliferative response in foals and adult horses infected with R. equi in comparison to other Vaps; both demonstrate the highest upregulation of the vap genes in macrophages in vitro (Hooper-McGrevy et al., 2003; Ren and Prescott, 2003; Jacks et al., 2007). The non-functional secreted protein VapD is highly induced due to macrophage related stress (Benoit et al., 2002; Ren and Prescott, 2003). VapG, a pH regulated protein that is secreted and may also be surface localized, is highly upregulated in foal macrophages, which is thought to be a response to stress within macrophages (Coulson et al., 2010). vapG has no effect on the intracellular replication of R. equi, and may play an important role in the initial host-pathogen interaction, or help disperse the pathogen from the lungs after a respiratory infection (Jain et al., 2003; Coulson et al., 2010). Expression of both vapD and vapG are induced through macrophage related stress and environmental signals, which are important pathophysiological factors in the virulence of R. equi (Benoit et al., 2002; Jacks et al., 2007; Coulson et al., 2010). The function of VapE, a secreted protein, and VapH are also unknown (Byrne et al, 2001). VapC, -D, and -E are low-molecular-mass immunogenic proteins that are thermoregulated and expressed at 37° C., in synchrony with VapA; coordinated regulation of multiple proteins is an important sign of virulence (Tan et al., 1994; Takai et al., 1996; Byrne et al., 1999; Byrne et al., 2001). The three vaps -F, -I, and -X, are non-functional proteins with unknown cellular locations (Meijer and Prescott, 2004; Cauchard et al., 2006; Polidori and Hass, 2006). vapl has been demonstrated to be unnecessary for the replication and survival in mouse macrophages and was observed to be upregulated by 6-fold when R. equi was grown in host cells (Polidori and Hass, 2006). Amino acid sequence analysis demonstrates that Vap proteins have high homology in their C-terminal domain, with the exclusion of VapF (Takai et al, 2000 (6840-6847). Although all yap genes are upregulated when R. equi is grown in equine macrophages, it was demonstrated that vapA, -D, and -G are greatly induced by macrophage related stress, and expressed at significantly higher levels in the lungs of infected foals (Benoit et al., 2002; Jacks et al., 2007; Coulson et al., 2010). When mutated, chromosomally encoded genes such as, nitrate reductase gene narG, isocitrate lyase gene aceA, high temperature requirement protein A gene, and the phoPR operon genes have demonstrated increased virulence or attenuation of R. equi, and are suggested to play a role in R. equi virulence in addition to the yap genes (Ren and Prescott, 2004; Wall et al., 2005; Pei et al., 2007). Furthermore, microarray and transcription network analysis demonstrate that plasmid encoded genes interact with housekeeping genes and cross-talk between the plasmid-chromosome genes, which is necessary for intracellular proliferation of R. equi (Letek et al, 2010). Table 2 summarizes the characteristics of each of the yap genes and their corresponding proteins.
TABLE 2Characteristics of R. equi vap genes and corresponding Vap proteins:KnownGeneR. equiCellularEncodedmethod ofimportantVapPlasmidlocation ofby ageneforproteinCategoryproteinpseudogeneregulationreplicationAvapA+surfacenoThermo/pHYesB*vapB+surfaceno——CvapA+secretednoThermo—DvapA+secretednoThermo/pH—EvapA+secretednoThermo—FvapA+notyes—NosecretedGvapA+secreted**noThermo/pH—HvapA+secretedno——IvapA+secretedyes——JvapB+————K1vapB+————K2vapB+————LvapB+————MvapB+————XvapA+—yes——Results expressed as a dash (—) indicate there is insufficent evidence from which to draw a reasonable conclusion.*VapB has only been observed in non-equine isolates.**May also be surface expressed.R. equi Vaccine Attempts
Recombinant VapA and VapC proteins have been used in several studies to test antibody levels against R. equiR. equi, and as immunogens to protect foals against an R. equiR. equi infection, or in donors for plasma production. Although a number of strategies have been studied and tested to develop an R. equiR. equi vaccine, a successful commercial vaccine has yet to be developed.
In the Chirino-Trejo 1987 study, protection of foals immunized orally against experimental R. equiR. equi was studied. Two strains of R. equiR. equi were used for immunization; one strain being a clinical isolate from a pneumonic foal (2523-85; CR +R) and the other a laboratory passaged Congo red negative (CR −R) variant of the same strain which differed in the absence of a dominant 17.5 kd and minor 15kd protein. This choice was made because in other facultative intracellular pathogens Congo red staining has been associated with virulence.
Two groups of three 1 to 3 week old foals were immunized administration of CR−R and CR+R via a stomach tube once a week for four weeks. The first group was immunized with 100 mL phosphate buffered saline containing 10^9-10^10 of CR+R. equiR. equi and the second group with 100 mL phosphate buffered saline containing 10^9-10^10 of CR −R. equiR. equi. Three non-immunized foals were used as controls. All foals were challenged three weeks after immunization, via aerosol infection with about 18 mL of about 10^10 of the pneumonic foal isolate. Additional aerosol exposure was performed on days 1, 2, 6, and 7 of the study (the first day of challenge being designated as day 0).
Foals in the control group were euthanized ten days after initial challenge day, along with one foal from each immunization group. The remaining foals were euthanized on day 14. The lungs of non-immunized foals showed signs of pneumonia, the lungs of one CR+ foal, sacrificed at the same day as the controls, showed areas of patchy consolidation which were significantly lower than that of the control foals. The lungs of the CR− foal, sacrificed at the same day as the controls, were congested and firm. The lungs of the CR+ and CR− foals euthanized on day 14 were congested but otherwise normal.
The procedure described for immunization appeared safe for foals but cannot be used on farms with endemic R. equiR. equi pneumonia problems. As described by Takai S et al. 1986, the multiplication of R. equiR. equi in intestine of foals causes dissemination of large numbers of virulent R. equiR. equi if administered by stomach tube. Therefore, natural infection will not be adequate to protectively immunize foals and an artificial immunization is required. Chirino-Trejo 1987 acknowledged that further studies must take place to identify the exact protective antigens and explore parental immunization.
Becú et al., 1997 performed a four-year study in Argentina to test the protection against R. equiR. equi pneumonia in foals via active and passive immunity. In field trial 1, between 700 and 1200 thoroughbred mares from 14 to 22 different farms were vaccinated with an R. equiR. equi vaccine containing many different antigens, including ‘equi factors’ and virulence associated protein VapA provided by Dr. J. F. Prescott. In the first two years (1992 and 1993), the mares were vaccinated at 45, 30, and 15 days prior to parturition. In the second two years (1994 and 1995), the mares were vaccinated at 30 and 15 days prior to parturition. From this group, 700 to 800 foals every year received only passive immunity.
In field trial 2, mares were vaccinated as described above. Foals with poor passive immunity were administered hyperimmune plasma (HIP) intravenously at 4 days of age. On two of the farms, all foals were administered HIP at 4 days of age. All 380 foals in this trial were given HIP at 25 days of age.
53 foals were used in field trial 3 and were divided up into three separate groups. The 33 foals in Group A were from vaccinated mares and all received HIP at 25 days of age. Five of these foals had poor passive transfer and were also administered HIP at 4 days of age. The ten foals in Group B were from non-vaccinated mares and were not administered HIP. These foals were immunized at 20, 30, and 40 days of age with 0.5 mL of the R. equiR. equi vaccine. The ten foals in Group C were from non-vaccinated mares and were neither given HIP nor vaccinated.
The immune response of all foals was tested until the foals were 90 days of age. The mortality from R. equiR. equi pneumonia in the foals from vaccinated dams dropped from an average of 3% to 1.2% in the 4 years of the study. The average mortality due to R. equiR. equi in foals administered HIP dropped from 5.8% to 0.2%. Active vaccination of foals from vaccinated mares on an enzootic farm at 20, 30, and 40 days of age did not protect them from mortality due to R. equiR. equi pneumonia. Becú et al., 1997 concluded that using their vaccine in a program of mare vaccination before parturition on enzootically affected farms significantly reduced morbidity and mortality. Furthermore, more complete control of enzootic pneumonia due to R. equiR. equi was achieved by administration of hyperimmune plasma to foals with low levels of antibodies to the vaccine antigens.
Hines et al. 2001 studied the hypothesis that horses develop an antigen-specific recall response after exposure to R. equiR. equi early in life, and can use this response to successfully clear R. equiR. equi bacteria when challenged. Twelve clinically healthy adult horses were used in this study over two consecutive summers. Representing a variety of breeds, the age of the horses ranged from 2 to 18 years. Three weekly bronchoalveolar lavages (BALs) were performed on each horse. During the first BAL procedure, the right lung of the horses was administered 2×10^7 R. equiR. equi strain ATCC 33701 per mL of PSB. The BAL procedure was repeated 7 and 14 days post-challenge, but no additional R. equiR. equi was administered.
Following each BAL procedure, the horses were placed in an isolated stall for one week after challenge and monitored for any changes in rectal temperature, respiration, or pulse. Any horse with a fever was further examined, including auscultation of the lungs using a re-breathing bag. Blood was taken from the horses vial jugular venipuncture on the day of each BAL procedure. Blood counts and fibrinogen levels were analyzed.
Using a pMal vector, Hines et al. 1996 constructed a recombinant VapA (rVapA) protein fused with maltose binding protein (MBP). The rVapA protein was used to study the lymphoproliferative response in BALF cells and PBMC from pre- and post-challenged horses. Prior to challenge with R. equiR. equi, most horses had minimal responses to rVapA protein; however, after challenge, the horses showed antigen-driven responses to rVap. From these findings, Hines et al. 1996 concluded that the administration of bacteria into the lungs led to the activation of memory T-cells with receptors for R. equiR. equi antigens. Support for further testing of VapA as an immunogen is associated with the recognition of VapA by T-cells of the lung.
A key study performed by Hooper-McGrevy et al. 2001, tried to determine whether immunity against R. equi conferred by hyperimmune plasma was mediated by antibodies against VapA and VapC. Twenty-eight 3-week old mixed breed pony foals ranging in age from 18 to 23 days were used in the study. 7 foals received 1 liter of commercially available hyperimmune plasma from whole-cell R. equi immunized donors (HIP) intravenously 24 hours prior to infection with R. equi. A second group of 7 foals received purified immunogloblulin against VapA and VapC 24 hours prior to infection. Foals were infected by administration of 25 ml of R. equi strain 103+ at a concentration of 5×10^7 CFU/ml into each of the major bronchi. 14 foals were used as controls in this study. Foals were euthanized 14 days after infection.
VapA and VapC recombinant proteins were produced by amplifying genes encoding VapA and VapC from the R. equi 103+ plasmid. Amplified products were digested and the digestions products were ligated to a similarly digested plasmid vector. Resultant plasmids were transformed into E. coli to allow for the in vitro production of recombinant VapA and VapC fused to glutathione-S-transferase (GST). Fusion proteins were purified using glutathione beads and VapA and VapC were cleaved from CST by use of thrombin.
Three adult horses were immunized intramuscularly 3 times at 2-week intervals with 1.5 mg each of VapA and VapC using 1 ml of aluminum hydroxide gel as the adjuvant. Three weeks after the third immunization, 4 liters of blood was collected from each horse and plasma was separated. Immunoglobulin was then precipitated from plasma, lyophilized and rehydrated in a volume of saline solution that resulted in a titer equivalent to that in the original plasma. Foals received 1 liter of this preparation intravenously 1 day prior to infection with R. equi. Heart rate, respiratory rate, and rectal temperature were recorded twice daily, and serum fibrinogen concentration and WBC count were determined every other day following infection.
Foals were euthanized 14 days following infection, and lung lesions and concentration of R. equi in lungs were assessed. The onset of clinical signs of pneumonia was significantly delayed in the HIP- and immunoglobulin-treated groups, compared with the untreated infected group. Moreover, pulmonary lesions were less severe in the treated groups, and significantly fewer R. equi organisms were cultured from the lungs of treated foals. Compared with untreated infected foals, severity of disease that developed following infection was reduced by administration of commercially available HIP as well as by administration of immunoglobulin purified from plasma of horses immunized with R. equi VapA and VapC.
Based on this study, Hooper-McGrevy concluded that immunoglobulin is the primary component of hyperimmune plasma that confers protection against R. equi-induced pneumonia in foals and that antibodies against R. equi VapA and VapC are protective. Significantly, Hooper-McGrevy observed that antibodies against VapA and VapC provided partial protection against R. equi induced pneumonia equivalent to that mediated by HIP. Thus, Hooper-McGrevy indicated that further studies are required to develop active immunization procedures that effectively protect foals from infection with R. equi. 
Lopez et al. 2002 set out to test whether antibodies to VapA would be expanded 14 days post challenge in adult horses. Lopez et al., 2002 developed a His-tagged recombinant VapA protein derived from ATCC 33701 to measure Vap protein levels from 12 adult horses challenged intrabrochially with R. equi. Horses were challenged with 2×10^7 R. equi strain ATCC 33701. 14 days post challenge, titers of all VapA antibody isotypes increased significantly, but the titers of IgGb and IgGa were dramatically enhanced following challenge. Lopez et al. 2002 concluded that an effective vaccine for foals must be designed to induce IgGb and/or the T-lymphocyte responses that production of this isotype reflects.
Hooper-McGrevy et al., 2003 developed recombinant GST-tagged Vap proteins for Vap proteins A, C, D, E, F, G and H derived from the R. equi strain 103+ plasmid. Proteins were produced in order to test the hypothesis that resistance of susceptibility to R. equi pneumonia in foals is associated with distinct IgG subtype-related antibody responses to the seven virulence-related Vap proteins and that in pneumonic foals the profile reflects a Th2-biased response whereas in healthy foals and adults, the profile reflects a Th1-biased response. 6 clinically normal pony foals from a farm with a history of R. equi infection in foals representing a clinically normal, R. equi exposed foal group, 6 clinically normal adult horses representing an R. equi immune group, and 8 foals with clinical R. equi pneumonia representing a nonimmune group, were used in this study. Hooper-McGrevy, et al., 2003 concluded that the higher the ratio of IgGa to IgGb subtype ratio and IgGa to IgGT ratio, the better the animals are protected against R. equi. 
Lopez, et al. 2003 set out to evaluate the immune response of foals to a DNA vaccine expressing a His-tagged recombinant VapA. Using the vapA gene amplified from the virulent R. equi strain 33701, Lopez, et al. 2003 designed a recombinant plasmid DNA containing the vapA gene (pVR1055vapA) and His-tagged recombinant VapA protein (rVapA). To characterize the primary immune response elicited by DNA immunization, 5 foals between 8 and 15 days of age (day 1) received 0.5 mg of pVR1055vapA intranasally. At day 14, foals received another 0.5 mg of pVR1055vapA intranasally. At day 30, foals received 0.1 mg of protein intranasally and 0.1 mg of protein containing 0.5 mls of RIBI adjuvant intradermally.
When examined at day 24 (10 days following the DNA boost), none of the foals had developed detectable antibodies against VapA. At day 45 (15 days following the protein boost), 2 of the 5 foals showed strong VapA-specific IgG antibody responses as measured by ELISA compared 20 with the control foals. Based on the observation that 2 foals mounted an immune response within 15 days of receiving a VapA protein boost, Lopez, et al. 2003 concluded that pVR1055vapA priming of naïve foals is suboptimal for the goal of protecting foals against R. equi and that a more profound protein boost, such as might occur with a low dose challenge with virulent R. equi organisms, may have been more successful at priming the foals. Lopez, et al. 2003 suggested that protecting foals against R. equi infection required priming and expanding lymphocytes within the first few weeks of life and proposed using a modified DNA based vapA vaccine whereby cytokine genes such as IL-12 and GM-CSF would be included in the vaccine. The most significant finding from this study was that the only immune response observed was after foals were immunized with the protein boost.
In a study aimed at evaluating serum IgG antibody levels and opsonizing activity in foals from pregnant mares immunized with either VapA or whole killed R. equi together with a water-based nanoparticle adjuvant, Cauchard, 2004 identified VapA as a candidate vaccine for immunizing pregnant mares resulting in passive antibody-mediated protection of foals. The whole formaldehyde-killed R. equi preparation (WKRE) was prepared from an isolate obtained from an infected foal from an endemically affected breeding farm in Normany, France. VapA was purified from R. equi strain 85F, which was obtained from a foal in the same geographic area. 24 mares were immunized with 1 mg of VapA protein at 9, 6 and 3 weeks before delivery, 8 mares were immunized with 10^9 bacteria at 24, 12 and 4 weeks before delivery. 15 mares served as controls and received adjuvant without purified protein or WKRE at 9, 6 and 3 weeks before delivery.
IgG antibodies were significantly higher in colostrum from the purified VapA immunized mares and WKRE immunized mares compared with the control mares, but no significant difference in titer between the VapA immunized mares and WKRE immunized mares was observed. After foals received colostrum, all foals exhibited R. equi antibody levels close to that of their mare. Foals born to WKRE immunized mares maintained the most stable antibody levels compared to foals born to VapA immunized mares and foals born to control mares.
Four of the 13 foals born to control mares developed proven R. equi pneumonia where all of the 23 foals born to VapA immunized mares and all of the 7 foals born to WKRE immunized mares remained free of R. equi induced pneumonia for the first 6 months of life. R. equi was confirmed by bacterial isolation from transtracheal aspirates, blood and feces.
Based on this study, in which environmental exposure was the only means of challenge, Cauchard et al. 2004 concluded that pregnant mare immunization with VapA protein antigen associated with a water-based nanoparticle adjuvant provides a more accessible, passive antibody-mediated protection than administration of hyperimmune plasma. This conclusion is based on the fact that serum opsonic activity in mares was higher in immunoglobulin from mares immunized with VapA than in either the control group or mares immunized with WKRE.
Taouji et al., 2004 tested synthetic VapA peptides (N15Y and V20S) representing B-cell epitope with two groups of adjuvants. Mice were immunized with 100 micrograms antigenic content twice at 2 week intervals. Mice were challenged with 2×10^6 R. equi ATCC 33701 4 weeks after the first vaccination and were euthanized and sampled 4 days after challenge. Mares ranging in age from 2 to 7 years were immunized subcutaneously with 0.5 mg antigenic content twice at 4 week intervals. Taouji et al. 2004 observed that the nanoparticle adjuvants elicicited the best immune response in comparison to metabolized oil adjuvant and that all adjuvants promoted anti-N15Y IgG2b and in a lesser extent, IgGa and IgGT. Additionally, antibodies to N15Y were detected in sera of immunized mares for 12 months. Taouji, et al. 2004 concluded that the N15Y peptide with the low-oil adjuvant was the most effective immunogen by elicicitng primary and memory immune response in mares, but that the efficiency of the N15Y peptide in combination with T-cell epitope would need to be further evaluated before challenge of foals.
Vanniasinkam et al., 2004 studied the immunogenicity of R. equi vaccines in a murine model for the potential use as vaccine candidates to protect against R. equi in foals. Four groups of five female BALB/c mice at 6-8 weeks of age were used in this study. Each group of mice was vaccinated separately with pcDNA3-Re1, His-tagged GroEL2, pcDNA3 vector, or 10^5 live virulent R. equi strain ATCC 33701. All animals were vaccinated three times, two weeks apart. Two weeks after the last immunization, all mice were challenged with an intravenous inoculation of 1.5×10^7 virulent R. equi strain ATCC 33701. Animals were symptom free a few days post-challenge, and bacterial clearance was measured 48-120 hours post-challenge. The protein-based vaccine elicited a mixed Th1/Th2 response, whereas the DNA vaccine elicited a predominantly Th1 biased immune response. When the vaccinated mice were challenged, neither vaccine enhanced bacterial clearance from the spleen or liver. Vanniasinkam et al., 2004 concluded that the co-administration of immunostimulatory molecules, such as IL-18 and IL-12, together with a pathogen-specific antigen-encoding DNA vaccine might be an option for enhancing a Th1 protective response. This approach may be useful for improvement of the Th1 type immunity observed.
Vanniasinkam et al. 2005 performed another study to determine the immune response elicited by R. equi VapA protein-based vaccines in a murine model. To test this, four groups of five 6-8 week old female BALE/c mice were vaccinated with pcDNA3-Re2, pcDNA3 vector, live R. equi, or His-tagged VapA protein. One group of mice received 5 μg of the murine cytokine IL-12 expression plasmid pORF-mIL12 co-injected with pcDNA3-Re2. All mice were vaccinated three times, two weeks apart. Two weeks after the last immunization, mice were challenged with an intravenous inoculation of 1.5×10^7 virulent R. equi strain ATCC 33701. Clearance of bacteria was measured 48-120 hours post-challenge. It was found that there was no significant difference in the rates of bacterial clearance from both the liver and spleen of mice with either the vapA-based DNA or protein vaccine compared with the control group. Overall, the results of this study suggest that VapA-based vaccines whether administered as DNA or recombinant protein vaccines do not enhance clearance of R. equi in the murine model, unlike the live R. equi. The efficacy of the live vaccine suggests that other R. equi antigens in addition to VapA may be required in order to induce a protective immune response to R. equi. Other strategies, including a heterologous prime/boost vaccination regiment, potent adjuvants, and viral vaccine carriers, could be employed to enhance the efficacy of the DNA vaccine used in this study.
Haghighi and Prescott, 2005 attempted to test the hypothesis that a DNA vaccine encoding vapA could induce a cell-mediated immune response to R. equi in mice and that the induction of this immunity confers protection against R. equi, and whether IL-12 would enhance this effect. A recombinant plasmid containing the vapA gene derived from the virulent strain ATCC 33701 (pcDNA3.1-vapA) was used in combination with a vector expressing IL-12 (pORF-mIL-12) as an adjuvant for pcDNA3.1-vapA. Mice were immunized with 100 micrograms of pcDNA3.1-vapA or 50 micrograms of pcDNA3.1-vapA and 50 micrograms of pORF-mIL-12. On day 21 after the first immunization, mice were challenged intravenously with 5×10^5 R. equi strain ATCC 33701 and were euthanized 4 days later. The DNA vaccine was effective in inducing a cellular immune response, and a significant increase in the VapA specific IgG2a to IgG1 ratio was observed. Using pORF-mIL-12 led to a significant increase in R. equi clearance from the liver, but a significant reduction in specific antibody titer. Haghighi and Prescott, 2005 concluded that DNA immunization using vapA and IL-12 has potential as a vaccine approach in foals and suggested that inclusion of other vap genes might also enhance the immune response.
As an extension of the Chirino-Trejo et al. 1987 study, Hooper-McGrevy et al. 2005 tried to determine whether oral immunization of foals with virulent R. equi strain ATCC 33701 within the first 2 weeks of life would induce protection by 3 weeks of age. This study also addressed whether or not it is possible to induce an effective immune response to R. equi in very young foals, given the difficulty to achieve efficacious vaccination of the neonate in the presence of maternal antibody.
The immunization schedule consisted of administration of 100 mL of phosphate-buffered saline (PBS), containing 1×10^8 CFU/mL of R. equi by stomach tube, 2 days after birth and again at 1 and 3 weeks of age. The control group contained 4 foals which were administered PBS only, by the same route. All foals were challenged at 3 weeks of age with 25 mL of R. equi containing 2×10^6 CFU/ml by intrabronchial inoculation into each of the major bronchi. After the challenge, the heart rate, respiratory rate and temperature of the foals were monitored twice daily. Blood was collected every other day for fibrinogen concentration, white blood cell count, and serology. Foals were euthanized via IV injection of pentobarbital sodium, 14 days after challenge (day of challenge being designated as day 0).
The non-immunized control foals remained healthy during the infection-immunization period, but developed signs of pneumonia by day 9 post-challenge. In comparison, the infection-immunized foals remained alert and healthy throughout the 2 week post-challenge. Assessment of the lungs postmortem showed that pneumonia occurred only in controls, but there was significant enlargement of lung-associated lymph nodes in foals from both groups. As a result of the pneumonia, the lungs of the control foals showed signs of obvious congestion and edema. The lungs of infection-immunized foals were free of lesions.
Analysis of antibody response was performed using Vap-specific ELISAs. The main difference in total antibody response between the control and immunized foal groups was for VapA and VapC. The anti-VapA and anti-VapC response in immunized foals was significantly greater, from day 0 through day 10 post-challenge and from day 2 through day 10, respectively. The total antibody response to the other Vap proteins did not differ.
Based on these results, Hooper-McGrevy concluded that future work should investigate the possibility of creating mutant strains of R. equi to reduce virulence but possess a majority of the immunogenic proteins such as VapA and VapC.
Mealey et al., 2007 attempted to induce a Type1/Th1 response in neonatal foals using a DNA-based R. equi vapA vaccine using recombinant equine IL-12 as a molecular adjuvant. Four foals under 8 days of age were immunized intradermally and intranasally with 1 mg of the DNA based vapA vaccine pVR1055vapA and 1 mg of IL-12 plasmid (day 1). On day 15, all foals received a second DNA vaccination. On day 30, all foals received a protein boost with 0.5 mg of purified recombinant VapA protein intradermally and 0.1 mg of purified recombinant VapA protein intranasally. Foals were challenged with R. equi ATCC 33701 virulent strain 2 weeks following the purified recombinant VapA protein boost. A challenge dose was 10^4 organisms per animal was used because the objective of this study was to evaluate immune responses with and without IL-12 DNA as an adjuvant and not to induce disease to evaluate protection for 2 weeks post-challenge. Mealey et al., 2007 was unable to detect a significant adjuvant effect in foals. Thus, the DNA based vapA vaccine pVR1055vapA was shown to be poorly immunogenic in foals.
Jacks et al., 2007 performed a study to determine the relative expression of R. equi vap genes and the lymphoproliferative responses in the lungs of infected foals. Five 7 to 10 day old foals and five 2 to 12 year old adult horses were used in the study. Transfer of passive immunity was confirmed in foals at 12 to 24 hours of age. Each foal was intrabronchially challenged with approximately 1×10^6 CFU of R. equi strain ATCC 33701, and each adult was intrabronchially challenged with approximately 1×10^7 CFU (2×10^4 CFU per kg of body weight). All of the animals were physically examined every day post-challenge for fifteen days, and their heart rate, respiratory rate, and temperature was recorded twice daily. At fifteen days post-challenge, all animals were euthanized, and bronchial lymph nodes (BLN) and cranioventral lung lobe tissue was aseptically collected.
To study the proliferative responses in the BLN and lobe tissue samples, GST-tagged recombinant VapA and VapC-H proteins were developed. Cells were separately incubated with no antigen (blank), 2 μg/mL of concanavalin A (positive control), 10 μg/mL of Corynebacterium pseudotuberculosis soluble antigens (negative control), or 50 μg/mL of each of the recombinant proteins. The lymphoproliferative response to VapA and VapC proteins in foals and adult horses was significantly greater than those to all other Vap proteins; however, Jacks et al. 2007 present data that indicates that vapA, vapD, and vapG are the most biologically relevant vap genes, due to their preferential induction during natural host infection.
Oliveira et. al. 2010 attempted to demonstrate the effects of immunization of mice with Salmonella enterica Typhimurium expressing the VapA antigen against R. equi infection. Five female BALB/c mice, 6 to 8 weeks of age, were intragastrically immunized with 1×10^9 CFU of S. enterica Typhimurium χ3987-pYA3137vapA. For the control group, five mice of the same type and age were intragastrically immunized with 1×10^9 CFU of S. enterica Typhimurium χ3987-pYA3137 and five mice were inoculated with 200 microliters of PBS only. The same pattern of immunization took place also 14 days post initial immunization.
All groups of mice were infected with a sublethal dose of virulent R. equi ATCC 33701, 14 days after the last immunization and euthanized. Based on analysis of samples of feces, spleen cells, and other organs Oliveira et al., 2010 concluded that VapA-immunization triggers mucosal-humoral response and systemic cell-mediated immunity, reflected by 5 fold higher IgA levels found in immunized mice compared to control mice. Additional analysis proved that R. equi antigen stimulates the production of Th1 Cytokines by spleen cells from mice vaccinated with S. enterica Typhimurium χ3987-pYA3137vapA. Additionally, vaccination with S. enterica Typhimurium χ3987-pYA3137vapA provided long term (duration of 5 months tested) protection of mice against R. equi infection. Based on the strong immune response of mice, Oliveira et al., 2010 suggested investigating immunization of foals against R. equi. 
Cauchard et al., 2011 created a Lactococcus lactis strain, LL-VapA, that expressed a His-tagged and S-Tagged VapA recombinant protein, and used the live bacterial strain LL-VapA to immunize mice. Mice were challenged with R. equi and it was shown that the LL-VapA strain helped induce an immune response in mice. However, the LL-VapA strain was not tested in horses.
Heuzenroeder, et al. 2009 used the VapA protein to design overlapping peptides, which provides an epitope map to be used in discovering potential vaccines for R. equi. 
Pei et al., 2007 generated an attenuated R. equi 103+ strain by disrupting the isocitrate lyase and cholesterol oxidase chromosomal genes (mutant). The goal was to assess the value of this strain for its immunizing ability. 6 ponies were immunized intrabronchially with 5×10^7 mutant R. equi at 7 days of age. At 21 days of age, foals were challenged with 5×10^7 virulent parent R. equi 103+. Foals were observed then euthanized 14 days post challenge. 3 foals remained unaffected by the mutant and were successfully immunized against infection with the parent R. equi strain. Pei et al. 2007 observed disease in a proportion of immunized foals, suggesting that the approach to producing double unmarked targeted mutations for use in immunization remains as a proof of principle.
A riboflavin auxotrophic live attenuated-vaccine stimulated an immune response but no protection against R. equi was observed when the foals were challenged (Lopez et al., 2008). A live attenuated-vaccine that targets the cholesterol catabolic gene cluster, necessary for R. equi virulence, was tested in foals and claimed to elicit an immune response against R. equi (van der Geize et al., 2011).